A three-dimensional model of CYP19 aromatase for structure-based drug design

Aromatase (CYP450_arom, CYP19) is an enzyme responsible for converting the aliphatic androgens androstenedione and testosterone to the aromatic estrogens estrone and estradiol, respectively. These endogenous hormones are a key factor in cancer tumor formation and proliferation through a cascade starting from estrogen binding to estrogen receptor. To interfere with the overproduction of estrogens especially in tumor tissue, it is possible to inhibit aromatase activity. This can be achieved using aromatase inhibitors. In order to design novel aromatase inhibitors, it is necessary to have an understanding of the active site of aromatase. As no crystal structure of the enzyme has yet been published, we built a homology model of aromatase using the first crystallized mammalian cytochrome enzyme, rabbit 21-progesterone hydroxylase 2C5, as a template structure. The initial model was validated with exhaustive molecular dynamics simulation with and without the natural substrate androstenedione. The resulting enzyme–substrate complex shows very good stability and only two of the residues are in disallowed regions in a Ramachandran plot.     

Dispersion of vendace eggs and larvae around potential nursery areas reveals their reproductive strategy

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Acidification of the intimal fluid: the perfect storm for atherogenesis

Atherosclerotic lesions are often hypoxic and exhibit elevated lactate concentrations and local acidification of the extracellular fluids. The acidification may be a consequence of the abundant accumulation of lipid-scavenging macrophages in the lesions. Activated macrophages have a very high energy demand and they preferentially use glycolysis for ATP synthesis even under normoxic conditions, resulting in enhanced local generation and secretion of lactate and protons. In this review, we summarize our current understanding of the effects of acidic extracellular pH on three key players in atherogenesis: macrophages, apoB-containing lipoproteins, and HDL particles. Acidic extracellular pH enhances receptor-mediated phagocytosis and antigen presentation by macrophages and, importantly, triggers the secretion of proinflammatory cytokines from macrophages through activation of the inflammasome pathway. Acidity enhances the proteolytic, lipolytic, and oxidative modifications of LDL and other apoB-containing lipoproteins, and strongly increases their affinity for proteoglycans, and may thus have major effects on their retention and the ensuing cellular responses in the arterial intima. Finally, the decrease in the expression of ABCA1 at acidic pH may compromise cholesterol clearance from atherosclerotic lesions. Taken together, acidic extracellular pH amplifies the proatherogenic and proinflammatory processes involved in atherogenesis.     

Evidence for an autumn downstream migration of Atlantic salmon Salmo salar (Linnaeus) and brown trout Salmo trutta (Linnaeus) parr to the Baltic Sea

In the eastern Baltic rivers, anadromous salmonid parr are known to smoltify and migrate to the sea from March until June, depending on latitude, climate and hydrological conditions. In this study, we present the first records of autumn descent of brown trout Salmo trutta and Atlantic salmon Salmo salar from the Baltic Sea Basin. Otolith microchemistry analyses revealed that these individuals hatched in freshwater and had migrated to the brackish water shortly prior to capture. The fish were collected in 2006, 2008, 2009 and 2013 from Eru Bay (surface salinity 4.5–6.5 ‰), Gulf of Finland. This relatively wide temporal range of observations indicates that the autumn descent of anadromous salmonids is not a random event. These results imply that autumn descent needs more consideration in the context of the effective stock management, assessment and restoration of Baltic salmonid populations and their habitats.     

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.     

Engagement of the NG2 proteoglycan triggers cell spreading via rac and p130cas

Cells that express the NG2 proteoglycan will spread on surfaces coated with monoclonal antibodies against this membrane-spanning protein. On surfaces coated with the N143 monoclonal antibody, this cell spreading occurs by extension of lamellipodia, suggesting that activation of the small GTPase rac is involved in the observed morphological change. Support for this hypothesis comes from the finding of increased levels of GTP-bound rac in cells spreading on N143-coated surfaces. Furthermore, lamellipodia extension is blocked by transfection of cells with the dominant negative rac construct N17rac, but not by transfection with N17cdc42. Formation of lamellipodia on the N143-coated surfaces is also inhibited by transfection of the dominant negative CasdeltaSD construct. This result implicates p130cas as an additional functional player in NG2-mediated cell spreading.